Immune recognition by most T lymphocytes occurs through surface interactions involving a specific T cell receptor (TCR) on the lymphocyte and an antigen on the target cell. Unlike antibodies, TCR do not recognize intact antigens. With few exceptions, they only recognize fragments of antigens, in the form of peptides, which are presented on the cell surface by molecules of the major histocompatibility complex (MHC). The ability of T cells to recognize antigen only when it is presented by MHC molecules is called “MHC restriction”. The reacting T cells are referred to as MHC-restricted lymphocytes. There are two classes of MHC molecules, class I and class II, which are functionally distinguished by the type of antigen they bind and the subset of T cells with which they interact. The dichotomy between class I and class II molecules relates to their different roles in T cell activation. Class I molecules present peptides to MHC-restricted CD8 positive cytotoxic T lymphocytes (CTL). These cells directly lyse target cells; therefore it is biologically appropriate that they recognize peptides derived from intracellular antigens that are presented by class I molecules. Moreover, since class I molecules are expressed by nearly all nucleated cells, CTL are able to recognize and destroy virtually any cell if it presents the appropriate MHC/peptide complex. On the other hand, class II molecules present peptides to CD4 positive T helper cells, the primary function of which is to secrete cytokines that promote activities of other lymphocytes, including B cells, macrophages and CTL. They play a dominant role in orchestrating an immune response.
MHC molecules are characterized by their highly polymorphic nature and many different variants have been defined which are encoded by different gene loci and alleles. Many of the amino acid variations that distinguish MHC alleles are localized in the binding groove where they modulate its shape and charge. Peptides capable of binding to a particular MHC molecule must fit to the configuration of that molecule's binding groove. Within the human population there are many different MHC alleles and an individual generally expresses six different class Ia molecules (encoded by the HLA-A, -B, C loci), each of which binds a different repertoire of peptides due to different amino acid composition of the peptide binding groove. In addition an individual has HLA alleles encoding a number of class Ib molecules, including HLA-E, F and G.
For MHC-restricted cells to respond to antigenic stimulation, NMC/peptide complexes must be expressed at the surface of a target cell and the T cells must express a cognate TCR. The specific ability of T-cells, to recognize specific antigens, which are presented via MHC molecules, was used in a variety of therapeutical approaches, e.g. in the antitumor therapy.
However, the known strategies do not take into consideration that the immune system has a substantial repertoire of non-MHC-restricted T-cells, which might be used for the elimination of tumor cells. This is partially due to the fact that attempts to use them in the anti-tumor therapy simply were not successful. Generally, the non-MHC-restricted type of T-lymphocytes combat tumors by their recognition of tumor cells, that occurs independently of MHC-peptide ligand expression.
Examples of cells which show non-MHC-restricted immune response are Lymphokine Activated Killer (LAK) cells. They represent mixtures of activated natural killer (NK) and non-MHC-restricted T cells that lyse various tumor cells as well as HLA class I negative target cells. Activated NK cells, like those present in LAK populations, are inhibited in their cytolytic function following interaction with MHC class I molecules. Regulation of LAK-derived T (LAK-T) cells has never been clarified.
The emerging understanding of the role of negative regulation of lymphocyte function through inhibitory receptor interactions with MHC class I molecules provides new insight into mechanisms that influence LAK function. While activating receptors such as natural cytotoxicity receptors are responsible for the induction of NK-mediated lysis, the cytolytic capacity of NK cells is ultimately determined by their inhibitory receptor expression. Natural killer cells (NK) are crucial for T cell activation in the present invention. Natural killer (NK) cells are a lineage of lymphoid cells, which lacks antigen-specific receptors, and NK cells are part of the innate immune system. These cells circulate in the blood as large lymphocytes with distinctive cytotoxic granules. These lymphocyte-like cells show important functions in innate immunity. They are able to recognize and kill some abnormal cells, for example tumor cells and virus-infected cells, and are thought to be important in the innate immune defence against intracellular pathogens. Although lacking antigen-specific receptors, they can detect and attack certain virus-infected cells.
Extensive analyses of different sources of NK cells have shown that essentially all human NK cells bear inhibitory receptors that interact with class I molecules and inhibit their cytotoxicity, thereby protecting normal somatic cells from NK-mediated attack (reviewed in(1-3). Two forms of inhibitory receptors are expressed by NK cells: the killer cell inhibitory receptors (KIR) belong to the immunoglobulin superfamily and interact with classical MHC class Ia (HLA-A, -B, -C) molecules whereas inhibitory receptors of the C-type lectin superfamily are composed of CD94/NKG2A heterodimers that bind non-classical MHC class Ib (HLA-E) molecules (4; 5). Through expression of class Ia and Ib molecules, nucleated cells bear two sets of ligands that can independently prevent their attack by NK cells bearing either of these inhibitory receptor types.
However, as stated above, the regulation of LAK-derived T (LAK-T) cells has never been clarified.
In recent years, new therapeutic approaches utilizing the immune system have been investigated for treatment of tumors that can not be eliminated by current chemotherapies or radiation therapies. The first immune therapies used systematic application of Interferons, either IFNα or FNγ, often in combination with IL-2 (6-9).
Rosenberg and colleagues were the first to explore the antitumor potential of LAK cells for immune therapy of cancer patients (10; 11). Following adoptive transfer of LAK cells into patients with advanced disease, some patients showed dramatic responses but many tumors failed to regress. Numerous animal studies and in vitro analyses of human LAK cells pinpointed activated NK cells as the major effector component mediating tumor regression, although weaker cytotoxic function was also found in the T cell fraction. However, clinical trials using LAK cells, applied either alone or in combination with high dose IL-2 to retain NK viability, failed to improve clinical efficacy. Furthermore, clinical benefits were severely limited by concurrent toxicity, particularly when IL-2 was coadministered with the LAK cells.
Further clinical trials studied the capacity of unseparated LAK cells to mediate antitumor activity in vivo following the adoptive transfer of large numbers of cells into patients with advanced disease (10; 12-20). While dramatic regression of some tumor lesions was observed, this occurred rarely, was short-lived and associated with high toxicity. In patients receiving systemic high dose IL-2 therapy, induction of LAK cells was observed in vivo and in vitro in most individuals, irrespective of their clinical responses, indicating that the efficacy of LAK cells was most likely regulated at the level of interaction between effector cells and tumor cells. The failure to identify the basis of LAK-mediated tumor regression, thereby allowing patients to be identified who might benefit from this therapy despite its associated toxicity, limited further clinical development.
This toxicity, together with the inability to understand why only some tumors regressed, led to the abandonment of non-MHC-restricted tumor cells, like LAK cells in favor of therapeutic strategies designed to adoptively transfer tumor-infiltrating lymphocytes or to induce MHC-restricted T cell responses. These newer strategies also show promise but, once again, tumor regression has only been observed in some patients. Interestingly, in several well-studied examples it was found that tumor variants emerged that showed partial or complete loss of MHC class I expression in patients who had generated strong class I-restricted CTL responses in vivo.
Thus, it appears that selective pressure by the CTL led to the emergence of tumor variants that no longer express the corresponding MHC-peptide complexes that are seen by the TCR of the CTL.
Extensive immunohistochemical studies of tumors have revealed a high prevalence of cells showing aberrant expression of HLA molecules, often limited to selected HLA allotypes. While such tumors still bind pan class I antibodies, like W6/32, their disturbed MHC expression might allow them to escape elimination by MHC-restricted CTL.
Therefore, it is the object of the present invention to provide an improved strategy for the treatment of tumors, which show a low, missing or aberrant expression of MHC class I molecules.
This problem is solved by the features set forth in the independent claims. Preferred embodiments of the present invention are detailed in the dependent claims.
The problems that are related to the therapies/compositions that have been used up to now are on the one hand due to the above described escape mechanism, which causes a reduced susceptibility of tumor cells for attack by MHC-restricted T-cells. On the other hand, as indicated above, therapies that involved non-MHC-restricted T-cells have not shown long lasting, reproducible and effective therapeutic results.
In recent experiments the inventors showed, that non MHC-restricted T-cells, e.g. LAK-T cells, like activated NK cells, are inhibited by interactions with HLA class I molecules. LAK-T cells lyse class I positive tumor cells but lysis is suppressed when tumor cells are stimulated with interferon-gamma to increase their class I expression. This inhibition of cytotoxicity is however reversed in the presence of class I-specific monoclonal antibody. HLA negative target cells can be partially protected from lysis by LAK-T cells following their transfection to express HLA class Ia or class Ib molecules. The principle of negative regulation by HLA molecules thus applies to non-MHC-restricted T-cells, such as LAK-T cells generated from tumor patients and healthy control donors. Although LAK-T cells are inhibited by class Ia and class Ib molecules, they do not express known NK inhibitory receptors. Apparently, they are negatively regulated through hitherto undefined inhibitory receptors. Experiments conducted by the inventors showed that these T-cells would be most effective in recognizing tumor cell variants that show low or disturbed MHC class I expression since their cytotoxic function could not be efficiently inhibited by interactions with class I molecules. These results have lead to a new approach, which allows an effective use of non-MHC restricted T-cells, e.g. LAK T-cells, in the therapy of tumor diseases by combating tumor variants showing low or aberrant expression of specific HLA allotypes.
Based on this research work, the use of therapeutic compositions containing immune cells, which attack tumor cells in a non-MHC-restricted way, in combination with MHC-restricted T-cells, provide a balanced selective pressure against emergence of tumor cell variants that would otherwise escape immune detection.
Generally, a therapeutic composition according to the present invention contains:                a) activated non-MHC-restricted T-cells and/or Natural Killer (NK) cells in combination with        b) MHC-restricted T-cells or        c) therapeutic agents, which induce immune responses of MHC-restricted T cells.        
These ingredients may also be contained in a kit of parts comprising one or more containers filled with one or more of the ingredients of the aforementioned composition of the invention. By means of this kit it is possible to separately or simultaneously administer those ingredients.